Beta-adrenergic agonists increase the myocardial contraction amplitude and enhance relaxation. Our studies examine the autonomic modulation of the calcium-myofilament interaction to determine whether it could account for enhanced twitch relaxation. In single cardiac myocytes we found that for a given amplitude of cytosolic calcium twitch amplitude was less in isoproterenol (Iso) than in control. In myocytes suspensions norepinephrine (NE, 10 micromolar) increased troponin-I phosphorylation fourfold and under similar conditions decreased twitch relaxation time in individual myocytes by 20%. After propranolol (1.0 micromolar) troponin slowly dephosphorylated (half time 16.9+1.7 min) but relaxation time fully returned to control within 5 min. Thus, the extent of troponin-I phosphorylation by NE is not directly proportional to its effect on relaxation time. In isolated, aequorin-injected ferret cardiac muscle the calcium tension relation was determined from the peak aequorin luminescence and peak tension measured across a broad range of bathing calcium in the presence and absence of acetylcholine (ACh, 1 micromolar) or Iso (1 micromolar) or both drugs. Ach shifted and relationship of peak tension to (peak) aequorin light leftward, suggesting an increase in myofilament calcium sensitivity, but did not alter twitch relaxation (t1/2R). Iso shifted the relationship of peak tension to peak aequorin light rightward and decreased t1/2R. Ach added to Iso abolished the Iso effect on peak tension-aequorin light relationship but did not reverse the effect of Iso to decrease t1/2R. Thus perturbations of the apparent myofilament calcium interaction in the intact muscle do not necessarily relate to twitch relaxation time.